LBH589 modulated lymphocyte secretion of cytokines.

<p>(<b>A</b>) ELISA for IFNgamma from supernatants of cultured cells. Left panel: PBMCs and L428 cells were cultured separately. L428 cells were co-cultured with PBMCs (middle panel) and CD3+ cells (right panel; t = 36 hours). In indirect co-cultures, cells were separated by the membrane. The ratios of effector to tumour cells were 1∶1 with PBMCs and 1∶2 with CD3+ cells. PBMC/L428 co-culture did not show significant results due to high intra-group variance. For CD3+ cells, a significant IFNgamma increase in direct co-culture compared with indirect co-culture (p = 0.0198) as well as a significant downregulation upon LBH589-treatment (p = 0.0192) was observed (two-way ANOVA analysis; solid lines indicate the comparison of indirect versus direct co-culture; dashed lines the effect of LBH589 [also in B) and C]). (<b>B</b>) ELISA for TNFalpha and settings like in A). For both, PBMC/L428 and CD3+/L428 co-cultures, the effect of indirect vs. direct co-culture was not significant (p = 0.6637 and p = 0.3187, respectively), however the LBH589 treatment increased the TNFalpha secretion significantly (middle and right subpanels; p = 0.0059 and 0.0006, respectively; two-way ANOVA analysis). (<b>C</b>) ELISA for TNFalpha release in lymphocyte/L428 co-cultures. The ratio of effector to tumour cells was 1∶4 with CD4+ and CD8+ cells each and 1∶10 in NK cell co-cultures. The ratio of effector to tumour cells was 1∶4 with CD4+ and CD8+ cells each; and 1∶10 in NK cell co-cultures. LBH589 increased significantly the TNFalpha secretion in co-cultures of L428 with CD4+, CD8+ and NK cells (p = 0.0001, p = 0.0017 and p = 0.0253, respectively). The direct versus indirect co-culture method showed a significant impact on CD4+ (p = 0.0006) and CD8+ lymphocytes (p = 0.0328; two-way ANOVA analysis). The ratio of effector to tumour cells was 1∶4 with CD4+ and CD8+ cells each and 1∶10 in NK cell co-cultures.</p

LBH589 enhanced cytotoxicity and worked synergistically with gemcitabine.

<p>(<b>A/B</b>) Killing assays in which target cells and PBMCs were pre-treated for 4 h with (<b>A</b>) 10 nM LBH or (<b>B</b>) 200 ng/ml TNFalpha. The panels show one representative experiment of three. The ratios of effector to target cells are indicated below the axis. In addition, the summary bar graph for the effector to target cell ratio 50∶1 of three independent experiments is given for both experimental settings (paired, two-sided t-test). (<b>C/D</b>) Combination regimens were tested with sublethal doses adjusted to each compound and cell line. L428 cells and L540 cells were treated with 10 nM LBH589 (LBH) and (<b>C</b>) Gemcitabine (GMZ; 500 ng/ml for L428 and 0.5 ng/ml for L540; n = 3) or (<b>D</b>) Everolismus (RAD; 5 µM for L428; 0.5 µM for L540; n = 3 each; paired, two-sided t-test).</p

video_1_Hodgkin Lymphoma-Derived Extracellular Vesicles Change the Secretome of Fibroblasts Toward a CAF Phenotype.mp4

<p>Secretion of extracellular vesicles (EVs) is a ubiquitous mechanism of intercellular communication based on the exchange of effector molecules, such as growth factors, cytokines, and nucleic acids. Recent studies identified tumor-derived EVs as central players in tumor progression and the establishment of the tumor microenvironment (TME). However, studies on EVs from classical Hodgkin lymphoma (cHL) are limited. The growth of malignant Hodgkin and Reed–Sternberg (HRS) cells depends on the TME, which is actively shaped by a complex interaction of HRS cells and stromal cells, such as fibroblasts and immune cells. HRS cells secrete cytokines and angiogenic factors thus recruiting and inducing the proliferation of surrounding cells to finally deploy an immunosuppressive TME. In this study, we aimed to investigate the role of tumor cell-derived EVs within this complex scenario. We observed that EVs collected from Hodgkin lymphoma (HL) cells were internalized by fibroblasts and triggered their migration capacity. EV-treated fibroblasts were characterized by an inflammatory phenotype and an upregulation of alpha-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts. Analysis of the secretome of EV-treated fibroblast revealed an enhanced release of pro-inflammatory cytokines (e.g., IL-1α, IL-6, and TNF-α), growth factors (G-CSF and GM-CSF), and pro-angiogenic factors such as VEGF. These soluble factors are known to promote HL progression. In line, ingenuity pathway analysis identified inflammatory pathways, including TNF-α/NF-κB-signaling, as key factors directing the EV-dependent phenotype changes in fibroblasts. Confirming the in vitro data, we demonstrated that EVs promote α-SMA expression in fibroblasts and the expression of proangiogenic factors using a xenograft HL model. Collectively, we demonstrate that HL EVs alter the phenotype of fibroblasts to support tumor growth, and thus shed light on the role of EVs for the establishment of the tumor-promoting TME in HL.</p

Role of BAT3 for iDCs and the effect of purified BAT3 on NK cell function.

<p>(A) Standard Europium release assay: Inhibition of NK-dependent lysis of iDCs in the presence of anti-BAT3 was significant (paired t-test, p-value = 0.008). (B) iDCs transfected with either control siRNA or BAT3 siRNA were co-incubated with NK cells for 4 hours at 37°C. The decrease of iDC lysis upon BAT3 down regulation was significant (p = 0.01). (C) Lysis of mature DCs upon pre-incubation with control antibodies, anti-HLA-ABC and/or anti-BAT3. (D) Co-culture of iDCs with activated NK cells at 5∶1 ratio (iDC∶NK) promotes the maturation of iDCs as shown by FACS analysis to detect expression of the maturation marker CD86. Inhibition of this effect is achieved by soluble purified BAT3. The y-axis represents the mean fluorescence intensity (MFI). (E) The lysis of Raji cells is inhibited by soluble BAT3 and anti-NKp30 compared to the control protein His BB4. The decrease of the lysis was significant (paired t-test, p value = 0.019). (F) NK cells were pre-stimulated with immobilized HisBB4 (control) and purified BAT3 prior a cytotoxicity assay with Raji cells as targets at different effector : target cell ratios. NK cells were derived from different donors for each experiment. Error bars for the lysis experiments represent standard deviation of three replicates. One representative experiment of four is shown.</p

Exosomal BAT3 regulates NK cell-function.

<p>(A) Western Blot to detect BAT3 in exosomal fractions upon over expression and depletion. Exosomes purified from untransfected cells (WT), BAT3-transfected cells (BAT3), control siRNA- transfected cells (si-c) and BAT3 siRNA- transfected cells were analysed by Western blotting to detect BAT3. (B) Exosomes were purified from media (PBS), untransfected 293T cells (wt), BAT3-transfected 293T cells (BAT3), control si-RNA (si-c) and BAT3 si-RNA (si-B) transfected 293T cells and used to stimulate NK cells. NK cell-supernatant was collected and used for a cytokine ELISA (TNF-α and IFN-γ). (C) NK cells were stimulated with exosomes derived from untreated iDCs (iDC-NHS exosomes) or upon heat shock (iDC-HS exosomes) for cytokine ELISA (left panels). NK cell-mediated cytokine release was estimated upon stimulation with exosomes derived from allogenic and autologous iDCs (right panels). Primary immune cells were derived from different donors for each experiment. The means of duplicates and the concentration (pg/ml) are indicated. One representative experiment of three is shown.</p

image_3_Hodgkin Lymphoma-Derived Extracellular Vesicles Change the Secretome of Fibroblasts Toward a CAF Phenotype.PDF

<p>Secretion of extracellular vesicles (EVs) is a ubiquitous mechanism of intercellular communication based on the exchange of effector molecules, such as growth factors, cytokines, and nucleic acids. Recent studies identified tumor-derived EVs as central players in tumor progression and the establishment of the tumor microenvironment (TME). However, studies on EVs from classical Hodgkin lymphoma (cHL) are limited. The growth of malignant Hodgkin and Reed–Sternberg (HRS) cells depends on the TME, which is actively shaped by a complex interaction of HRS cells and stromal cells, such as fibroblasts and immune cells. HRS cells secrete cytokines and angiogenic factors thus recruiting and inducing the proliferation of surrounding cells to finally deploy an immunosuppressive TME. In this study, we aimed to investigate the role of tumor cell-derived EVs within this complex scenario. We observed that EVs collected from Hodgkin lymphoma (HL) cells were internalized by fibroblasts and triggered their migration capacity. EV-treated fibroblasts were characterized by an inflammatory phenotype and an upregulation of alpha-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts. Analysis of the secretome of EV-treated fibroblast revealed an enhanced release of pro-inflammatory cytokines (e.g., IL-1α, IL-6, and TNF-α), growth factors (G-CSF and GM-CSF), and pro-angiogenic factors such as VEGF. These soluble factors are known to promote HL progression. In line, ingenuity pathway analysis identified inflammatory pathways, including TNF-α/NF-κB-signaling, as key factors directing the EV-dependent phenotype changes in fibroblasts. Confirming the in vitro data, we demonstrated that EVs promote α-SMA expression in fibroblasts and the expression of proangiogenic factors using a xenograft HL model. Collectively, we demonstrate that HL EVs alter the phenotype of fibroblasts to support tumor growth, and thus shed light on the role of EVs for the establishment of the tumor-promoting TME in HL.</p

image_4_Hodgkin Lymphoma-Derived Extracellular Vesicles Change the Secretome of Fibroblasts Toward a CAF Phenotype.PDF

<p>Secretion of extracellular vesicles (EVs) is a ubiquitous mechanism of intercellular communication based on the exchange of effector molecules, such as growth factors, cytokines, and nucleic acids. Recent studies identified tumor-derived EVs as central players in tumor progression and the establishment of the tumor microenvironment (TME). However, studies on EVs from classical Hodgkin lymphoma (cHL) are limited. The growth of malignant Hodgkin and Reed–Sternberg (HRS) cells depends on the TME, which is actively shaped by a complex interaction of HRS cells and stromal cells, such as fibroblasts and immune cells. HRS cells secrete cytokines and angiogenic factors thus recruiting and inducing the proliferation of surrounding cells to finally deploy an immunosuppressive TME. In this study, we aimed to investigate the role of tumor cell-derived EVs within this complex scenario. We observed that EVs collected from Hodgkin lymphoma (HL) cells were internalized by fibroblasts and triggered their migration capacity. EV-treated fibroblasts were characterized by an inflammatory phenotype and an upregulation of alpha-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts. Analysis of the secretome of EV-treated fibroblast revealed an enhanced release of pro-inflammatory cytokines (e.g., IL-1α, IL-6, and TNF-α), growth factors (G-CSF and GM-CSF), and pro-angiogenic factors such as VEGF. These soluble factors are known to promote HL progression. In line, ingenuity pathway analysis identified inflammatory pathways, including TNF-α/NF-κB-signaling, as key factors directing the EV-dependent phenotype changes in fibroblasts. Confirming the in vitro data, we demonstrated that EVs promote α-SMA expression in fibroblasts and the expression of proangiogenic factors using a xenograft HL model. Collectively, we demonstrate that HL EVs alter the phenotype of fibroblasts to support tumor growth, and thus shed light on the role of EVs for the establishment of the tumor-promoting TME in HL.</p

video_2_Hodgkin Lymphoma-Derived Extracellular Vesicles Change the Secretome of Fibroblasts Toward a CAF Phenotype.mp4

<p>Secretion of extracellular vesicles (EVs) is a ubiquitous mechanism of intercellular communication based on the exchange of effector molecules, such as growth factors, cytokines, and nucleic acids. Recent studies identified tumor-derived EVs as central players in tumor progression and the establishment of the tumor microenvironment (TME). However, studies on EVs from classical Hodgkin lymphoma (cHL) are limited. The growth of malignant Hodgkin and Reed–Sternberg (HRS) cells depends on the TME, which is actively shaped by a complex interaction of HRS cells and stromal cells, such as fibroblasts and immune cells. HRS cells secrete cytokines and angiogenic factors thus recruiting and inducing the proliferation of surrounding cells to finally deploy an immunosuppressive TME. In this study, we aimed to investigate the role of tumor cell-derived EVs within this complex scenario. We observed that EVs collected from Hodgkin lymphoma (HL) cells were internalized by fibroblasts and triggered their migration capacity. EV-treated fibroblasts were characterized by an inflammatory phenotype and an upregulation of alpha-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts. Analysis of the secretome of EV-treated fibroblast revealed an enhanced release of pro-inflammatory cytokines (e.g., IL-1α, IL-6, and TNF-α), growth factors (G-CSF and GM-CSF), and pro-angiogenic factors such as VEGF. These soluble factors are known to promote HL progression. In line, ingenuity pathway analysis identified inflammatory pathways, including TNF-α/NF-κB-signaling, as key factors directing the EV-dependent phenotype changes in fibroblasts. Confirming the in vitro data, we demonstrated that EVs promote α-SMA expression in fibroblasts and the expression of proangiogenic factors using a xenograft HL model. Collectively, we demonstrate that HL EVs alter the phenotype of fibroblasts to support tumor growth, and thus shed light on the role of EVs for the establishment of the tumor-promoting TME in HL.</p

table_1_Hodgkin Lymphoma-Derived Extracellular Vesicles Change the Secretome of Fibroblasts Toward a CAF Phenotype.xlsx

<p>Secretion of extracellular vesicles (EVs) is a ubiquitous mechanism of intercellular communication based on the exchange of effector molecules, such as growth factors, cytokines, and nucleic acids. Recent studies identified tumor-derived EVs as central players in tumor progression and the establishment of the tumor microenvironment (TME). However, studies on EVs from classical Hodgkin lymphoma (cHL) are limited. The growth of malignant Hodgkin and Reed–Sternberg (HRS) cells depends on the TME, which is actively shaped by a complex interaction of HRS cells and stromal cells, such as fibroblasts and immune cells. HRS cells secrete cytokines and angiogenic factors thus recruiting and inducing the proliferation of surrounding cells to finally deploy an immunosuppressive TME. In this study, we aimed to investigate the role of tumor cell-derived EVs within this complex scenario. We observed that EVs collected from Hodgkin lymphoma (HL) cells were internalized by fibroblasts and triggered their migration capacity. EV-treated fibroblasts were characterized by an inflammatory phenotype and an upregulation of alpha-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts. Analysis of the secretome of EV-treated fibroblast revealed an enhanced release of pro-inflammatory cytokines (e.g., IL-1α, IL-6, and TNF-α), growth factors (G-CSF and GM-CSF), and pro-angiogenic factors such as VEGF. These soluble factors are known to promote HL progression. In line, ingenuity pathway analysis identified inflammatory pathways, including TNF-α/NF-κB-signaling, as key factors directing the EV-dependent phenotype changes in fibroblasts. Confirming the in vitro data, we demonstrated that EVs promote α-SMA expression in fibroblasts and the expression of proangiogenic factors using a xenograft HL model. Collectively, we demonstrate that HL EVs alter the phenotype of fibroblasts to support tumor growth, and thus shed light on the role of EVs for the establishment of the tumor-promoting TME in HL.</p

Bio-chemical characterization of the released BAT3.

<p>(A) Detection of BAT3 expression on exosomes by electron microscopy {left panel: gold antibody control (140000×) and right panel: exosomes stained with anti-BAT3 antibody (140000×)}. (B) Western blotting to detect BAT3, Hsp70, Lamp-2 and CD9 in exosomal fractions (30 µg) and lysate (10 µg) of 293T cells and iDCs. (C,D) FACS analysis to detect BAT3 and various surface markers on exosomes, that were purified from iDCs (C) or 293T cells (D) that were immobilized to latex beads. Grey background represents isotype control. (E) FACS analysis of exosomes derived from control transfected (wt) or BAT3-transfected (BAT3) 293T cells revealed over-expression of BAT3 on the exosomal surface. Specific binding of anti-BAT3, NKp30-Ig and NKp46-Ig was detectable. Grey histograms: background (secondary antibody) staining of beads coated with exosomes. (F) Western blot analysis demonstrates that the enhanced secretion of BAT3 into the supernatant obtained from tumor cells (293T) when treated with heat shock (HS, lane: 3) or left untreated (UT, lane: 2). Lanes 4 and 5 demonstrate the co-immunoprecipitation of BAT3 by using either a polyclonal BAT3 antibody (4^th lane) or a monoclonal Ab against Hsp70 (5^th lane). The western blot is stained for BAT3. Lane 1 (M) indicates the marker.</p